Integrated In Vitro and In Silico Characterization of 5‐Hydroxyferulic Acid: Antioxidant, Anti‐Inflammatory, Anti‐Hemolytic, and Cytotoxic Potential

ABSTRACT

In the pursuit of novel antioxidant and anti‐inflammatory agents, we investigated 5‐hydroxyferulic acid (5‐OHFA), a hydroxylated derivative of the well‐known phenolic compound ferulic acid (FA). This study aimed to determine whether structural modification enhances the biological activity of FA. To this end, both compounds were subjected to a series of in vitro antioxidant assays (DPPH, ABTS, FRAP, and Fe(II)‐chelating) and anti‐inflammatory evaluations, complemented by in silico predictions. 5‐OHFA consistently exhibited superior antioxidant capacity, with significantly lower IC₅₀ values than FA based on literature‐reported values: 11.89 ± 0.20 versus 66 ± 2.3 µM (DPPH), 9.51 ± 0.15 versus 183.08 ± 2.30 µM (ABTS), 5.94 ± 0.09 versus 4.73 ± 0.14 µM (FRAP), and 36.31 ± 1.36 versus 270.27 ± 1.14 µM (Fe²⁺ chelation). It also demonstrated stronger anti‐inflammatory potential in protein denaturation assays using egg albumin and bovine serum albumin (BSA). Although 5‐OHFA showed slightly greater hemolytic activity (IC₅₀ = 23.78 ± 1.48 µM) than FA, based on literature‐reported values (37.64 ± 2.01 µM), both remained within biologically acceptable limits. In silico analyses using SwissADME and ProTox III supported the experimental findings, predicting good oral bioavailability, high gastrointestinal absorption, mild blood–brain barrier permeability, and no significant toxicity for 5‐OHFA. Molecular docking studies further revealed stronger binding affinities of 5‐OHFA to key oxidative and inflammatory targets, including NADPH oxidase (2CDU), xanthine oxidase (1FIQ), 5‐lipoxygenase (3O8Y), cyclooxygenase‐2 (3LN1), myeloperoxidase (1DNU), and the EGFR enzyme's active pocket (1M17). Overall, the data suggest that 5‐OHFA possesses enhanced bioactivity relative to its parent compound FA, supporting its potential as a promising multifunctional candidate for pharmaceutical or nutraceutical development.

1. Introduction

Natural products have historically represented a cornerstone in drug discovery, largely due to their immense structural diversity and wide‐ranging biological activities. They continue to serve as lead compounds for the development of new pharmacological agents, particularly in the context of chronic diseases where conventional therapies often face limitations related to efficacy, safety, or resistance. Among these natural compounds, phenolic acids occupy a prominent place due to their ability to modulate key biochemical pathways implicated in oxidative stress and inflammation.

A particularly well‐studied phenolic acid is ferulic acid (FA), chemically known as 4‐hydroxy‐3‐methoxycinnamic acid. This hydroxycinnamic acid is ubiquitously found in plant cell walls, especially in grains, fruits, and vegetables. Numerous studies have highlighted its antioxidant, anti‐inflammatory, anti‐cancer, and antimicrobial activities, which stem primarily from its free radical scavenging capacity, metal chelating ability, and the capacity to modulate pro‐oxidant and proinflammatory enzymes [1–4].

Driven by its broad pharmacological potential, recent research has focused on developing structural analogs of FA with improved physicochemical and biological properties [5–7]. Among them, 5‐hydroxyferulic acid (5‐OHFA)—a hydroxylated derivative featuring an additional hydroxyl group at the 5‐position of the aromatic ring—has emerged as a promising compound. This modification is hypothesized to improve its hydrophilicity, electron‐donating capacity, and radical stabilization potential, thereby enhancing its antioxidant activity [8]. Despite these promising chemical attributes, very little is known about the pharmacological profile of 5‐OHFA, especially regarding its anti‐inflammatory and anti‐hemolytic activities or its ability to modulate enzymatic targets involved in oxidative and inflammatory cascades.

Most of the available data concerning 5‐OHFA remains theoretical or chemically descriptive. To date, no comprehensive evaluation of its biological activities, particularly its capacity to interact with key enzymes such as NADPH oxidase (NOX), xanthine oxidase (XO), cyclooxygenase‐2 (COX‐2), 5‐lipoxygenase (5‐LOX), myeloperoxidase (MPO), and epidermal growth factor receptor (EGFR), has been conducted either in silico or experimentally.

Oxidative stress and chronic inflammation are interconnected pathological processes implicated in a broad spectrum of noncommunicable diseases, including cardiovascular diseases, neurodegenerative disorders (e.g., Alzheimer's and Parkinson's), metabolic syndromes (e.g., diabetes), and even certain viral infections [9, 10]. A hallmark of these conditions is the excessive production of reactive oxygen species (ROS) and pro‐inflammatory mediators, often driven by specific enzymatic systems.

Among the major enzymatic contributors to oxidative stress and inflammation, NOX plays a central role by catalyzing the production of superoxide anion (${\mathrm{O}}_2^{-}$), a primary precursor of various ROS. Similarly, XO contributes to oxidative stress by generating both superoxide and hydrogen peroxide during purine catabolism, a process directly implicated in hyperuricemia and vascular oxidative injury. On the inflammatory front, COX‐2 and 5‐LOX are pivotal enzymes in the biosynthesis of prostaglandins and leukotrienes, respectively—two families of lipid‐derived mediators that orchestrate key aspects of inflammation, including vasodilation, chemotaxis, and pain sensitization. Another critical player is MPO, a heme‐containing enzyme abundantly stored in neutrophil azurophilic granules, which catalyzes the formation of hypochlorous acid (HOCl) from hydrogen peroxide and chloride ions. HOCl is a highly reactive oxidant that, while bactericidal, also contributes to tissue damage and chronic inflammatory pathology when produced in excess [11–13].

Given the complexity of signaling pathways involving EGFR and other interconnected molecular targets, multi‐target modulation has emerged as a promising strategy in modern drug development. By simultaneously targeting multiple enzymes, this approach aims to enhance therapeutic efficacy while minimizing toxicity and the risk of resistance. It is especially relevant in diseases marked by cytokine storms or intricate feedback mechanisms, where single‐target therapies often prove insufficient [14, 15].

The EGFR, a member of the receptor tyrosine kinase (RTK) family, plays a pivotal role in the regulation of cell proliferation, differentiation, and apoptosis. Its overexpression and aberrant activation are strongly implicated in the development and progression of several malignancies, including colorectal, lung, breast cancers, and glioblastoma. In these contexts, EGFR contributes to uncontrolled cellular proliferation, evasion of apoptosis, and enhanced metastatic potential [16, 17].

Phenolic acids, due to their structural features—aromatic rings with hydroxyl and methoxy substitutions—can act as dual or triple inhibitors against COX, LOX, and NOX pathways. Studies have shown that FA and its analogs can effectively modulate these enzymes, reinforcing their therapeutic interest as multifunctional agents [14, 15, 18, 19].

In recent years, the combination of in silico and in vitro methodologies has become increasingly valuable in natural product research. Molecular docking, in particular, offers mechanistic insight into the interaction between small molecules and target proteins, predicting binding modes, affinity, and key residues involved. For phenolic acids, this tool helps elucidate their inhibitory mechanisms toward oxidative and inflammatory enzymes such as XO, NOX, COX‐2, 5‐LOX, and MPO [20].

By predicting the most relevant interactions, in silico screening can guide experimental design, reducing time and resources. In this study, molecular docking was used to predict the binding affinity of 5‐OHFA to the above‐mentioned targets, and complementary in vitro assays were conducted to validate these interactions. Antioxidant activity was evaluated using 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS), FRAP, and ferrous ion chelation assays, while protein denaturation and red blood cell hemolysis inhibition assays were used to assess anti‐inflammatory and membrane‐protective effects, respectively.

Despite the promising structural properties of 5‐OHFA, its biological efficacy and mechanisms of action have not been adequately characterized. To address this gap, the present study undertakes a comprehensive bibliographic and mechanistic investigation of 5‐OHFA's potential as a multi‐target antioxidant and anti‐inflammatory agent.

By integrating in silico predictions with data from functional in vitro assays, this work aims to: (1) Elucidate the molecular interactions of 5‐OHFA with key oxidative and inflammatory enzymes, such as NOX, XO, COX‐2, 5‐LOX, and MPO, using molecular docking simulations; (2) assess the biological relevance of hydroxylation at the 5‐position by comparing the antioxidant, anti‐inflammatory, and anti‐hemolytic activities of 5‐OHFA to those of its parent compound, FA; (3) support the therapeutic potential of 5‐OHFA as a multifunctional natural compound targeting oxidative stress and chronic inflammation‐related disorders; and (4) evaluate the cytotoxicity of 5‐OHFA on five human cell lines—HCT116 (colorectal carcinoma), LS174T (colorectal adenocarcinoma), WM266‐4 (metastatic melanoma), MCF‐7 (breast adenocarcinoma), and HEK293 (nontumoral embryonic kidney cells)—to determine its biosafety profile and its relevance for pharmaceutical and nutraceutical applications.

2. Materials and Methods

2.1. Chemicals and Reagents

5‐OHFA (Cat. no. XYZ123, purity ≥ 98%) was purchased from Rare Chemicals GmbH (Kiel, Germany) and is of synthetic origin according to the supplier. Standard analytical reagents used throughout the study included DPPH, ABTS, butylated hydroxytoluene (BHT), potassium persulfate, potassium ferricyanide, ferric chloride, ferrous chloride, trichloroacetic acid, ferrozine, ethylenediaminetetraacetic acid (EDTA), diclofenac sodium (DCF), bovine serum albumin (BSA), ethanol, methanol, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) and crystal violet (Cat. no. C3886), all obtained from Sigma‐Aldrich (Deisenhofen, Germany) or Fluka. Dulbecco's modified Eagle medium (DMEM) and RPMI‐1640 medium were obtained from Sigma‐Aldrich (Merck, Darmstadt, Germany). Fetal calf serum (FCS) was obtained from Gibco (Langley, OK, USA). Phosphate‐buffered saline (PBS) and other routine culture supplements were also obtained from Sigma‐Aldrich (Saint‐Quentin, France). All chemicals were of analytical grade and used without further purification.

2.2. Antioxidant Activity Assays of 5‐OHFA

2.2.1. DPPH Radical Scavenging Assay

The free radical scavenging activity of 5‐OHFA was evaluated using the DPPH assay, as previously described by Chokri et al. [21], with minor modifications. A series of 5‐OHFA concentrations ranging from 2 to 24 µM was accurately prepared. For each concentration, 1 mL of 0.078 mM DPPH solution (prepared in ethanol) was added, and the mixture was vortexed (Vortex mixer, Fisher Scientific, Waltham, MA, USA) before being incubated in the dark at room temperature for 30 min. The absorbance of the resulting solution was then measured at 517 nm using a UV–Vis spectrophotometer (Techcomp S1020). BHT was used as the standard antioxidant for comparative purposes. The test was performed in triplicate, and the percentage of DPPH radical inhibition was calculated using the following equation:

$$\mathrm{Inhibition}\left(\%\right)=\left[\left(\mathrm{Ab}{\mathrm{s}}_{\mathrm{control}}-\mathrm{Ab}{\mathrm{s}}_{\mathrm{sample}}\right)\right]/\left(\mathrm{Ab}{\mathrm{s}}_{\mathrm{control}}\right)]\times 100$$

where Abs_control is the absorbance of the DPPH solution without the sample (blank) and Abs_sample is the absorbance in the presence of 5‐OHFA.

A linear regression analysis was performed to determine the IC₅₀ value, which represents the concentration of the compound required to inhibit 50% of the DPPH radicals. A lower IC₅₀ indicates a stronger radical scavenging potential, thus reflecting the antioxidant efficacy of 5‐OHFA.

2.2.2. ABTS Radical Cation Scavenging Assay

The antioxidant potential of 5‐OHFA was further assessed using the ABTS radical scavenging assay, following the protocol described by Chokri et al. [21], with slight modifications. The ABTS radical cation (ABTS•⁺) was generated by mixing 7 mM ABTS solution (pH 7.4) with 2.5 mM potassium persulfate, followed by incubation in the dark at room temperature for 16 h. Before analysis, the resulting solution was diluted to achieve an absorbance of 0.70 ± 0.02 at 734 nm.

For the assay, 20 µL of 5‐OHFA sample, at different concentrations (2–24 µM), was added to 1 mL of the freshly prepared ABTS•⁺ solution. The mixture was manually agitated and incubated at room temperature for 10 min. Absorbance was then measured at 734 nm using a UV/Vis spectrophotometer (Techcomp S1020). BHT was used as a reference antioxidant. The percentage of ABTS radical scavenging was calculated using the same formula as in the DPPH assay:

$$\mathrm{Inhibition}\left(\%\right)=\left[\left(\mathrm{Ab}{\mathrm{s}}_{\mathrm{control}}-\mathrm{Ab}{\mathrm{s}}_{\mathrm{sample}}\right)/\mathrm{Ab}{\mathrm{s}}_{\mathrm{control}}\right]\times 100$$

where Abs_control is the absorbance of the ABTS solution without sample (blank) and Abs_sample is the absorbance in the presence of 5‐OHFA.

2.2.3. Ferric‐Reducing Antioxidant Power Assay

The ferric‐reducing antioxidant power (FRAP) assay is a widely adopted method to assess the electron‐donating ability of antioxidants, reflecting their potential to reduce Fe^3⁺ (ferric) ions to Fe²⁺ (ferrous) ions. This reduction process leads to the formation of a blue‐colored Prussian blue complex, which can be quantified by measuring absorbance at 700 nm. The assay is conducted under mildly acidic conditions (pH 6.6), which enhances the solubility of iron and favors electron transfer reactions.

In this study, the FRAP assay was carried out based on the method described by Chokri et al. [21], with slight modifications. A reaction mixture was prepared by combining 1.25 mL of 0.2 M phosphate buffer (pH 6.6), 1.25 mL of 1% potassium ferricyanide [K₃Fe(CN)₆], and 1 mL of 5‐OHFA at various concentrations (ranging from 2 to 60 µM). The mixture was incubated at 50°C for 20 min to allow the reduction of ferricyanide to ferrocyanide. After incubation, the reaction was stopped by adding 1.25 mL of 10% trichloroacetic acid, and the mixture was centrifuged (Eppendorf centrifuge, Eppendorf, Hamburg, Germany) at 4000 rpm for 10 min to remove any precipitate. An aliquot of the supernatant (1.25 mL) was then mixed with 1.25 mL of distilled water and 0.25 mL of freshly prepared 0.2% ferric chloride (FeCl₃). The resulting mixture was incubated in the dark for 10 min, and the absorbance was measured at 700 nm using a UV–Vis spectrophotometer (Techcomp S1020). This assay measured the formation of the ferric iron ion complex (Prussian blue), which is indicative of reducing potential. The greater the absorbance measured, the stronger the reducing power of the sample.

2.2.4. Ferrous Ion (Fe²⁺)‐Chelating Assay

The chelating ability of 5‐OHFA toward ferrous ions (Fe²⁺) was assessed using the ferrozine colorimetric method, following the protocol of Chokri et al. [21], with minor modifications. This assay is based on the ability of ferrozine to form a stable purple‐colored complex with Fe²⁺, which can be quantitatively measured at 562 nm. In the presence of a chelating agent, complex formation is inhibited, resulting in decreased absorbance proportional to the degree of metal ion binding.

Briefly, 0.4 mL of 5‐OHFA at varying concentrations (1–15 µM) was mixed with 0.05 mL of 2 mM FeCl₂ solution. After 1 min of incubation at room temperature to allow initial coordination, 0.2 mL of 5 mM ferrozine solution was added to the mixture. The total volume was adjusted to 4 mL. After vigorous shaking, the mixture was left to equilibrate at room temperature for 10 min in the dark. Absorbance was measured at 562 nm using a UV–Vis spectrophotometer (Techcomp S1020). A control sample containing all reagents except the test compound was used to define 100% complex formation. EDTA served as a positive control and reference chelator. The chelation percentage was calculated using the formula:

$$\%\mathrm{Chelation}=\left[1-\left(\mathrm{Ab}{\mathrm{s}}_{\mathrm{sample}}-\mathrm{Ab}{\mathrm{s}}_{\mathrm{control}}\right)\right]\times 100$$

where Abs_sample is the absorbance of the tested compound and Abs_control is the absorbance of the control. The results are expressed as the IC₅₀ concentration, where 50% inhibition of the Fe²⁺–ferrozine complex was obtained.

This combination of DPPH, ABTS, FRAP, and Fe(II)‐chelating assays provides a comprehensive evaluation of the antioxidant potential of 5‐OHFA, highlighting its ability to scavenge free radicals and its potential therapeutic application.

2.3. In Vitro Anti‐Inflammatory Activity

2.3.1. BSA Denaturation Assay

The anti‐inflammatory potential of 5‐OHFA was evaluated using the BSA denaturation method, as described by Ben Attia et al. [22], with slight modifications. Protein denaturation is a well‐established marker of inflammation, and the ability to prevent heat‐induced denaturation of BSA reflects potential anti‐inflammatory effects. Briefly, various concentrations of 5‐OHFA (0.625–25 µg/mL) were prepared and mixed with 1 mL of 0.2% (w/v) BSA solution. The mixtures were incubated at 37°C for 20 min to promote interaction between the compound and the protein. This was followed by heating at 57°C for 3 min to induce denaturation. After cooling to room temperature, 2.5 mL of phosphate buffer (pH 6.4) was added to each tube. The absorbance was measured at 255 nm using a UV–Visible spectrophotometer (Techcomp S1020). A control sample, containing BSA and buffer without the test compound, represented 100% denaturation. DCF was used as a reference anti‐inflammatory drug under the same experimental conditions. The percentage inhibition of protein denaturation was calculated using the following formula:

$$\mathrm{Inhibition}\left(\%\right)=\left[\left(\mathrm{Ab}{\mathrm{s}}_{\mathrm{control}}-\mathrm{Ab}{\mathrm{s}}_{\mathrm{sample}}\right)/\mathrm{Ab}{\mathrm{s}}_{\mathrm{sample}}\right]\times 100$$

where Abs_control indicates the denatured BSA without 5‐OHFA, while Abs_sample refers to the amount of BSA incubated with 5‐OHFA or diclofenac. The findings represent the average of three replicates.

This method provides a simple yet reliable in vitro model to assess the anti‐inflammatory activity of natural or synthetic agents by quantifying their ability to protect proteins from thermal denaturation.

2.3.2. Egg Albumin Denaturation Assay

The anti‐inflammatory potential of 5‐OHFA was assessed using the heat‐induced egg albumin denaturation method, as described by Ben Attia et al. [22] with slight modifications. In this assay, protein denaturation—a key marker of inflammatory damage—was induced by thermal stress, and the ability of 5‐OHFA to inhibit this process was evaluated. A 5 mL reaction mixture was prepared by combining 0.2 mL of fresh egg albumin, 2.8 mL of PBS (pH 6.4), and 2 mL of 5‐OHFA at concentrations ranging from 25 to 200 µg/mL. DCF, a standard anti‐inflammatory drug, was used under the same conditions as a reference. A double volume of distilled water served as the control. The samples were incubated at 37 ± 2°C for 15 min, followed by heating at 72°C for 5 min to induce protein denaturation. After cooling to room temperature, the absorbance was measured at 660 nm using a UV–Vis spectrophotometer (Techcomp S1020). The percentage of protein denaturation inhibition was calculated using the formula:

$$\mathrm{Inhibition}\left(\%\right)=\left[\left(\mathrm{Ab}{\mathrm{s}}_{\mathrm{control}}-\mathrm{Ab}{\mathrm{s}}_{\mathrm{sample}}\right)/\mathrm{Ab}{\mathrm{s}}_{\mathrm{sample}}\right]\times 100$$

where Abs_control indicates the denatured albumin without 5‐OHFA, while Abs_sample refers to the albumin incubated with 5‐OHFA or diclofenac. All experiments were performed in triplicate. This assay provides complementary evidence of the protective effects of 5‐OHFA against protein destabilization associated with inflammatory responses.

2.4. Erythrocyte Heat‐Induced Hemolysis

The protective effect of 5‐OHFA against heat‐induced erythrocyte hemolysis was evaluated following the method of Espinoza‐Culupú et al. [23], with slight modifications. Fresh blood was collected from healthy mice into heparinized tubes and centrifuged at 3000 rpm for 5 min to separate erythrocytes. The red blood cells were washed three times with 0.9% saline solution and then diluted to obtain a 10% (v/v) erythrocyte suspension. For the assay, 0.05 mL of erythrocyte suspension was mixed with 0.05 mL of 5‐OHFA at the desired concentration (0.5–50 mM) and 2.9 mL of phosphate buffer (pH 7.4). The mixture was gently stirred and incubated at 54°C for 20 min to induce membrane destabilization. After incubation, the tubes were centrifuged at 2500 rpm for 3 min, and the absorbance of the resulting supernatant was measured at 550 nm using a UV–Vis spectrophotometer. A phosphate buffer solution lacking the test compound was used as the control to represent 100% hemolysis. The percentage of hemolysis inhibition was calculated using the formula:

$$\mathrm{Hemolysis}\left(\%\right)=\left(A_2/A_1\right)\times 100$$

where A ₁ is the absorbance of the control and A ₂ is that of the treated sample. All experiments were conducted in triplicate. This assay offers insight into the membrane‐stabilizing and cytoprotective properties of 5‐OHFA under oxidative stress conditions.

2.5. Cytotoxicity Assessment

2.5.1. Cell Culture

Four human cancer cell lines were used in this study: HCT116 (colorectal carcinoma, ATCC CCL‐247), LS174T (colorectal carcinoma goblet cells, ATCC LS174T), and WM266‐4 (metastatic melanoma, ATCC CRL‐1676), which were cultured in RPMI‐1640 medium (Gibco), and MCF‐7 (breast carcinoma, ATCC HTB‐22), which was cultured in DMEM (Gibco). All media were supplemented with 10% fetal bovine serum (FBS; Gibco), 2 mM l‐glutamine (Sigma‐Aldrich, St. Louis, MO, USA), and 1% penicillin–streptomycin solution (Gibco). Cultures were maintained at 37°C in a humidified incubator under 5% CO₂ atmosphere.

2.5.2. MTT Assay

The cytotoxic potential of 5‐OHFA was evaluated using the MTT assay, as described by Belaiba et al. [24], with minor modifications. Five cell lines were tested: MCF‐7, HEK293, HCT116 and LS174T, and WM266‐4. Cells were seeded into 96‐well microplates (TPP, Switzerland) at a density of 3 × 10⁴ cells per well in 100 µL of culture medium. MCF‐7 and HEK293 cells were cultured in DMEM, while RPMI‐1640 medium was used for HCT116, LS174T, and WM266‐4. After 24 h of incubation to allow cell adherence in a CO₂ incubator (Thermo Fisher Scientific, Waltham, MA, USA), the cells were treated with 100 µM 5‐OHFA for 48 h. Following treatment, the medium was removed and replaced with 50 µL of MTT (Sigma‐Aldrich, Steinheim, Germany) solution (3 mg/mL in PBS). Plates were incubated at 37°C for 40 min to allow formazan crystal formation. The supernatant was discarded, and the crystals were solubilized in 50 µL of DMSO. Absorbance was measured at 605 nm using a microplate reader (Multiskan GO, F1‐01620, Thermo Fisher Scientific, Vantaa, Finland). Cell viability was expressed as a percentage of growth inhibition relative to untreated controls using the formula:

$$\mathrm{Inhibition}\left(\%\right)=\left[\left(\mathrm{Ab}{\mathrm{s}}_{\mathrm{control}}-\mathrm{Ab}{\mathrm{s}}_{\mathrm{sample}}\right)/\mathrm{Ab}{\mathrm{s}}_{\mathrm{control}}\right]\times 100$$

All experiments were performed in triplicate and repeated in three independent assays. Tamoxifen (100 µM) was used as a positive control to compare the anti‐proliferative efficacy of 5‐OHFA.

2.6. In Silico Analysis

2.6.1. ADMET Prediction

The pharmacokinetic properties, drug‐likeness, and toxicity profile of 5‐OHFA were evaluated using in silico tools. SwissADME (https://www.swissadme.ch/; accessed in 2024) was employed to predict absorption, distribution, metabolism, and excretion (ADME) parameters, alongside key physico‐chemical descriptors. Drug‐likeness was assessed based on compliance with Lipinski's rule of five, Veber's rule, and other criteria such as molecular weight, hydrogen bond donors/acceptors, topological polar surface area (TPSA), and lipophilicity (logP) [25]. The BOILED‐Egg predictive model was utilized to evaluate passive gastro‐intestinal absorption and blood–brain barrier (BBB) permeability. Oral bioavailability was further explored using bioavailability radar plots, incorporating six critical parameters: lipophilicity (LIPO), size (SIZE), polarity (POLAR), solubility (INSOLU), flexibility (FLEX), and saturation (INSATU). In parallel, the ProTox‐III webserver (https://tox‐new.charite.de/; accessed in 2024) was used to estimate the compound's toxicity profile, including predicted LD₅₀ values, hepatotoxicity, organ toxicity, BBB penetration, cytochrome P450 (CYP2D6) inhibition, and plasma membrane binding potential. This in silico analysis provided valuable insight into the pharmacokinetic suitability and potential safety of 5‐OHFA as a therapeutic candidate.

2.6.2. Molecular Docking

Molecular docking simulations were conducted to explore the binding affinities and interaction profiles of 5‐OHFA with key enzymes implicated in oxidative stress, inflammation, and hemolysis. Target proteins included NOX (PDB: 2CDU), XO (PDB: 1FIQ), COX‐2 (PDB: 3LN1), 5‐LOX (PDB: 3O8Y), MPO (PDB: 1DNU), and EGFR protein (PDB: 1M17) with their crystal structures retrieved from the RCSB Protein Data Bank (https://www.rcsb.org/; accessed in 2024). Protein structures were prepared using Discovery Studio 4.0 by removing water molecules, adding polar hydrogens, and assigning Kollman charges. Ligand and receptor structures were converted to pdbqt format using AutoDock Tools 1.5.6. Docking simulations were performed with AutoDock Vina, with the grid box centered at the catalytic site of each enzyme (dimensions: 60 × 60 × 60 Å; grid spacing: 0.375 Å). The best docking poses were selected based on binding affinity scores. Ligand–protein interactions were further visualized and analyzed in two and three dimensions using Discovery Studio Visualizer 2017, allowing for detailed assessment of hydrogen bonds, hydrophobic contacts, and active site occupancy.

3. Results

3.1. DPPH Free Radical Scavenging Activity

The exploration of antioxidant compounds, particularly those derived from natural sources, has garnered considerable attention due to their capacity to neutralize free radicals and prevent diseases associated with oxidative stress. Among the most widely used methods for evaluating antioxidant activity is the DPPH assay, which assesses the ability of compounds to scavenge stable free radicals.

In the present study, the antioxidant potential of 5‐OHFA was evaluated using the DPPH assay. The results revealed that 5‐OHFA exhibited strong antioxidant activity, with an IC₅₀ value of 11.89 ± 0.20 µM (Table 1). In comparison, FA has been reported to exhibit lower antioxidant capacity, with an IC₅₀ value of 66 ± 2.30 µM [26]. Based on these literature‐reported values, 5‐OHFA appears significantly more effective in scavenging free radicals, highlighting its enhanced antioxidant potential.

TABLE 1.

The IC₅₀ values of the antioxidant activities of 5‐hydroxyferulic acid and FA.

	IC50 DPPH	IC50 ABTS	IC50 FRAP	IC50 Chelating activity	References
Unit	µM	µM	µM	µM
5‐OHFA	11.89 ± 0.20	9.51 ± 0.15	5.94 ± 0.09	36.31 ± 1.36	This study
FA	66 ± 2.30	183.08 ± 2.30	4.73 ± 0.14	270.27 ± 1.14	[26, 30, 31, 49]

Note: Data for 5‐OHFA are expressed as mean ± SD from three independent experiments. Data for FA are extracted from previously published studies [26, 30, 31, 48]. As the FA values originate from different literature sources, no statistical comparison was performed between 5‐OHFA and FA.

Abbreviations: ABTS, 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulphonic acid); DPPH, 2,2‐diphenyl‐1‐picrylhydrazyl; FRAP, ferric‐reducing antioxidant power.

Furthermore, the antioxidant efficacy of 5‐OHFA was found to be concentration‐dependent, as increasing concentrations led to progressively higher DPPH radical scavenging rates. At the highest tested concentration of 23.78 µM, 5‐OHFA achieved a scavenging rate of 99.70%, as presented in Figure 1a. This clear dose–response relationship highlights the potent free radical scavenging capacity of 5‐OHFA and supports its potential as a promising natural antioxidant.

FIGURE 1.

Antioxidant activities of 5‐OHFA assessed through four in vitro assays: (a) DPPH radical scavenging activity, (b) ABTS radical scavenging activity, (c) ferric‐reducing antioxidant power (FRAP), and (d) ferrous ion chelating activity. Results are expressed as mean ± standard deviation (SD) from three independent experiments.

3.2. ABTS⁺ Scavenging Activity

The ABTS assay is a widely recognized method for evaluating the free radical‐scavenging capacity of antioxidants. In the present study, 5‐OHFA exhibited remarkable antioxidant activity, with an IC₅₀ value of 9.51 ± 0.15 µM, indicating a strong and concentration‐dependent ability to neutralize ABTS⁺ radicals (Table 1). This performance markedly exceeds that of FA and isoferulic acid (IFA), which have reported IC₅₀ values of 15.86 and 44.64 µM, respectively [27]. Furthermore, [28] reported a much higher IC₅₀ for FA (183.08 µM), and Halpani et al. [26] observed an IC₅₀ of 12 µM, further reinforcing the superior antioxidant capacity of 5‐OHFA under comparable experimental conditions.

Importantly, the scavenging response of 5‐OHFA exhibited a clear dose‐dependent trend. As the concentration increased from 2 to 18 µM, the percentage of ABTS⁺ radical inhibition (% DO) rose correspondingly from 12% to 74%. For instance, at a concentration of 4.75 µM, 5‐OHFA achieved 25% inhibition, while at 17 µM, the inhibition reached 74%. These values, which can be found in Figure 1b, reflect a progressive and consistent increase in antioxidant activity with increasing concentration. The experimental data fit well to an exponential regression model (y = 5.5155e^{0.9422 x} ), with a strong correlation coefficient (R ² = 0.9862) as shown in Figure 1b, confirming the robustness of the observed dose–response relationship. At the highest tested concentrations, 5‐OHFA achieved near‐maximal radical scavenging, further demonstrating its high efficiency and potential as a natural antioxidant agent.

3.3. Fe²⁺‐Chelating Assay

The Fe²⁺‐chelating activity of 5‐OHFA was evaluated based on its ability to disrupt iron–ferrozine complexes, providing further insight into its antioxidant mechanisms (Table 1). The assay revealed a clear concentration‐dependent chelating effect, with chelation values increasing from 1% at 2 µM to 62% at 55 µM (Figure 1c). At the highest tested concentration, 5‐OHFA reached a maximum chelation efficiency of 63%. The calculated IC₅₀ was 36.31 ± 1.36 µM, reflecting a relatively strong affinity for iron ions and a significant potential to limit oxidative processes catalyzed by free iron. In comparison, FA exhibited a much weaker chelating activity, with an IC₅₀ of 270.27 ± 1.14 µM under similar experimental conditions [29, 30]. These results highlight the markedly superior metal‐chelating capacity of 5‐OHFA and underscore the enhanced functional properties conferred by hydroxylation of the parent compound.

3.4. FRAP Assay

The FRAP assay was employed to assess the reducing capacity of 5‐OHFA, an important indicator of its antioxidant potential. As shown in Table 1, 5‐OHFA demonstrated strong reducing power activity, with an IC₅₀ of 5.94 ± 0.09 µM. In contrast, literature‐reported IC₅₀ values for FA and trans‐FA indicate lower iron‐binding efficacy, at 4.73 µM and 5.06 mM, respectively [28, 31]. These comparisons suggest that 5‐OHFA has a markedly enhanced reducing capacity relative to its structural analogs. As shown in Figure 1c, 5‐OHFA exhibited a clear concentration‐dependent increase in ferric‐reducing activity, as evidenced by the progressive rise in absorbance at 700 nm. Specifically, absorbance values increased from 0.84 at 1 µM to 1.895 at 14 µM, reflecting a strong positive correlation between concentration and reducing power. This linear dose–response relationship confirms that 5‐OHFA acts as an effective electron donor, contributing to its ability to neutralize oxidative species through redox‐based mechanisms. These findings reinforce the multifunctional antioxidant behavior of 5‐OHFA and support its potential application in managing oxidative stress through both radical scavenging and metal‐reducing pathways.

These results highlight the superior antioxidant capacity of 5‐OHFA, particularly its enhanced ability to chelate ferrous ions. This stronger iron‐binding activity positions 5‐OHFA as a more potent antioxidant compared to FA, which demonstrates relatively lower chelation efficiency. The comparison reinforces the consistent and significant role of 5‐OHFA in mitigating oxidative stress through effective modulation of transition metal ions. Such metal‐reducing capabilities are critical for preventing iron‐catalyzed oxidative damage, thereby supporting the therapeutic potential of 5‐OHFA in oxidative stress‐related disorders.

3.5. Anti‐Inflammatory Potential of 5‐OHFA: In Vitro Evaluation

The anti‐inflammatory potential of 5‐OHFA was systematically assessed through two in vitro protein denaturation models: BSA and egg albumin denaturation assays (Figure 2).

FIGURE 2.

Anti‐inflammatory activity of 5‐hydroxyferulic acid (5‐OHFA) evaluated through protein denaturation assays: (a) Inhibition of bovine serum albumin (BSA) denaturation; (b) inhibition of egg albumin denaturation in the presence of diclofenac as a standard drug. Results are presented as mean ± SD from three independent experiments.

In the BSA denaturation assay, the inhibitory activity of 5‐OHFA was compared to that of diclofenac, a reference nonsteroidal anti‐inflammatory drug (NSAID). At a concentration of 25 µg/mL, 5‐OHFA inhibited protein denaturation by 70%, while diclofenac achieved a higher inhibition rate of 97% under the same conditions. The IC₅₀ value for 5‐OHFA was calculated to be 15 µg/mL, whereas that of diclofenac was 2.5 µg/mL, indicating that 5‐OHFA possesses significant anti‐inflammatory activity, although it is less potent than the reference drug in this model (Figure 2a).

Similarly, in the egg albumin denaturation assay, 5‐OHFA demonstrated an inhibition rate of 73% at 200 µg/mL, while diclofenac showed 88% inhibition under the same conditions. The IC₅₀ values for 5‐OHFA and diclofenac were 125 and 112.5 µg/mL, respectively. The relatively small (Figure 2b) difference in IC₅₀ values highlights a comparable anti‐inflammatory effect between the two compounds in this assay.

Collectively, these findings confirm the dose‐dependent anti‐inflammatory efficacy of 5‐OHFA and support its potential as a promising natural compound for the development of novel anti‐inflammatory agents.

3.6. Heat‐Induced Hemolysis

The integrity and lifespan of erythrocytes are critically influenced by their metabolic activity and vulnerability to oxidative stress [32]. In the present study, the protective effect of 5‐OHFA against heat‐induced hemolysis of erythrocytes was evaluated across a concentration range of 0.5 to 50 mM. As shown in Figure 3, only mild hemolytic activity was observed at the highest tested concentration (47.5 mM), suggesting a favorable safety profile. The half‐maximal inhibitory concentration (IC₅₀) of 5‐OHFA for the prevention of hemolysis was calculated to be 23.78 ± 1.48 mM, indicating a notable capacity to preserve erythrocyte 50% integrity under thermal oxidative stress. This protective effect is likely attributable to the radical‐scavenging properties of 5‐OHFA, which may mitigate lipid peroxidation and maintain the structural stability of erythrocyte membranes.

FIGURE 3.

Effect of different concentrations of 5‐hydroxyferulic acid (5‐OHFA) on heat‐induced hemolysis: comparison of percentage hemolysis (■) and inhibition of hemolysis (■), with representative images of treated erythrocyte suspensions.

3.7. In Vitro Anti‐cancer Activity of 5‐OHFA

The anti‐cancer activity of 5‐OHFA was evaluated using the MTT assay against four human cancer cell lines after 48 h of exposure: colorectal carcinoma cells (HCT116), goblet cells derived from colorectal carcinoma (LS‐174T), metastatic melanoma cells (WM266‐4), and breast cancer cells (MCF‐7). Tamoxifen (100 µM) served as the positive control.

As summarized in Table 2, 5‐OHFA displayed notable inhibitory activity against HCT116 cells, achieving close to 50% growth inhibition, indicating promising anti‐proliferative effects. In contrast, the compound did not inhibit the growth of LS‐174T cells at the same concentration.

TABLE 2.

Comparative cytotoxic effects of 5‐OHFA and tamoxifen on various human cancer cell lines.

Samples	HCT‐116	MCF‐7	WM266‐4	LS174T
5‐OHFA	49.79 ± 1.46	16.73 ± 2.46	28.28 ± 2.08	0
Tamoxifen	65.2 ± 3.07	79.56 ± 2.17	76 ± 1.96	69.61 ± 8.17

For the MCF‐7 cell line, 5‐OHFA showed moderate inhibition of 16.73 ± 2.46%, substantially lower than the 79.56 ± 2.17% inhibition observed with tamoxifen. Regarding the WM266‐4 melanoma cells, the inhibition rate reached 28.28 ± 2.08%, still significantly lower than tamoxifen's 76 ± 1.96% inhibition.

3.8. In Silico Analysis

To explore the pharmacokinetic parameters, predicted biological targets, and anti‐inflammatory potential of 5‐OHFA, its chemical structure (Figure 4a) was employed in canonical SMILES format: [H]OC(═O)C[H]═C(/[H])C1═CC(O[H])═C(O[H])C(OC)═C1. This representation enabled comprehensive in silico analyses, including Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) profiling, to assess the compound's pharmacokinetic behavior and drug‐likeness. For molecular docking studies, the three‐dimensional (3D) structure of 5‐OHFA was retrieved from the PubChem database (PubChem CID: 446834) in PDB format, which provides an accurate spatial conformation for computational modeling. The structure was accessed on February 11, 2024, via the following link: 5‐Hydroxyferulic acid | C10H10O5 | CID 446834 – PubChem (nih.gov) [33]. This 3D model was used to simulate the molecular interactions of 5‐OHFA with relevant protein targets, thereby supporting its potential as a bioactive therapeutic candidate.

FIGURE 4.

(a) Chemical structure of 5‐hydroxyferulic acid, oxygen in red and nitrogen in blue. (b) Predicted BOILED‐Egg diagram. (c) Bioavailability radar from the SwissADME web tool; the pink area shows the optimal range of particular properties. LIPO (lipophilicity), SIZE (molecular weight), POLAR (polarity), INSOLU (insolubility in water), INSATU (unsaturation), and FLEX (flexibility). (d) Protein target class distribution for 5‐hydroxyferulic acid via SwissTargetPrediction.

3.8.1. ADMET Profiling

To evaluate the potential of 5‐OHFA as a promising drug candidate, comprehensive in silico ADMET profiling was performed (Table 3). Key pharmacokinetic parameters were predicted, including the partition coefficient (log P), TPSA, molecular weight, number of hydrogen bond donors and acceptors, rotatable bonds, and compliance with Lipinski's “rule of five.” The predictions were conducted using established computational tools, namely, Molinspiration (v2022.08), SwissADME, and ProTox‐II.

TABLE 3.

Molecular properties of 5‐hydroxyferulic acid.

Molecular properties	Molinspiration	SwissADME
Molecular weight	210.19 g/mol	210.18 g/mol
Number heavy atoms	15	15
Aromatic heavy atoms	6	6
Fraction Csp3	N/A	0.10
Number rotatable bonds	3	3
Number of H‐bond acceptors/nON	5	5
Number of H‐bond donors/nOHNH	3	3
Molar Refractivity	N/A	53.65
TPSA (Å2)	86.99	86.99
miLogP	0.96	1.10
Lipinski violation	0	0
Solubility log S	N/A	−1.95
Consensus log P o/w	N/A	1.01

The results revealed favorable pharmacokinetic characteristics. The log p value indicated adequate lipophilicity, supporting efficient permeability across biological membranes. Similarly, the TPSA value suggested good oral bioavailability, aligning with properties desirable for oral drug delivery (Table 3). The molecular weight of 210.19 g/mol, along with fewer than 10 hydrogen bond acceptors and fewer than 5 hydrogen bond donors, confirms adherence to Lipinski's rule, further highlighting its drug‐like nature. In addition, the moderate number of rotatable bonds indicates a suitable degree of molecular flexibility, which is often advantageous for optimal target binding.

Toxicological predictions using the ProTox‐II platform estimated an LD₅₀ value of 1772 mg/kg, corresponding to a low‐to‐moderate toxicity classification. No significant hepato‐toxicity was predicted, suggesting a favorable safety profile. Nonetheless, these findings must be substantiated through experimental validation.

The BOILED‐Egg model analysis (Figure 4b) predicted high gastro‐intestinal absorption for 5‐OHFA but limited passive BBB permeability, suggesting its suitability for systemic but not central nervous system (CNS) targets. Moreover, the bioavailability radar (Figure 4c) showed that most physico‐chemical properties of 5‐OHFA fall within the optimal range (pink zone), with only a slight deviation in the unsaturation (INSATU) index, which may have a marginal effect on overall oral bioavailability.

In summary, in silico profiling supports the drug‐likeness of 5‐OHFA, demonstrating compliance with established pharmacokinetic criteria, favorable absorption and safety profiles, and minimal CNS penetration. These promising attributes warrant further in vitro and in vivo investigations to confirm its pharmacological potential and therapeutic applicability.

3.8.2. Target and Anti‐Inflammatory Prediction

A comprehensive in silico investigation was performed to predict the molecular targets and anti‐inflammatory potential of 5‐OHFA. The predictions revealed a diverse range of protein interactions, highlighting the compound's therapeutic versatility. Specifically, 5‐OHFA showed predicted binding affinities to various classes of proteins, including lyases, proteases, oxidoreductases, and membrane receptors, which are involved in critical physiological and pathological processes such as inflammation, oxidative stress, and immune regulation (Figure 4d).

The prediction of activity spectra for substances (PASS) analysis further supported the potential anti‐inflammatory activity of 5‐OHFA, indicating a high probability (Pa > 0.7) for anti‐inflammatory, antioxidant, and radical scavenging activities. These predictions align with its known structural features, particularly the phenolic hydroxyl groups, which are commonly associated with ROS scavenging and inflammatory enzyme inhibition.

Notably, the compound demonstrated an affinity for key anti‐inflammatory targets such as COX‐2 and 5‐LOX, as well as for enzymes implicated in oxidative stress, including XO and NOX. These molecular interactions were confirmed through molecular docking studies, which revealed strong binding scores and favorable interactions with the active sites of these enzymes, supporting their functional inhibition (Table 4).

TABLE 4.

Details of the interacting residues and binding affinities of 5‐hydroxyferulic acid and ferulic acid.

	5‐Hydroxyferulic acid		Ferulic acid
	Binding affinity (kcal/mol)	Residues H‐bonding	Binding affinity (kcal/mol)	Residues H‐bonding
NADPH oxidase	−6.1	THR9, HIS79	−5.9	SER326, ILE160
Xanthine oxidase	−7.0	THR1083, ALA1079, SER1080	−5.6	VAL764, LYS792
Cyclooxygenase‐2	−5.8	GLN360, ASN361, GLY211, ASN361	−5.7	ASN361, ASN361, ARG362
5‐Lipoxygenase	−6.1	TYR470, GLN549	−6.2	ARG370, THR545
Myeloperoxidase	−5.0	ASN189, MET190, SER191, VAL199	−4.8	ALA166, GLU180
EGFR	−5.9	Lys721, Leu764, Met769	−5.8	ASN676, ALA678, LEU736

Moreover, network pharmacology analysis and docking simulations collectively emphasized the multifaceted mechanism of action of 5‐OHFA. This compound appears to act through the modulation of multiple signaling pathways rather than a single‐target effect. Such polypharmacology is especially advantageous for treating complex diseases like inflammation, where multiple biochemical cascades are simultaneously involved.

Together, these results suggest that the biological activity of 5‐OHFA stems from its ability to engage a broad range of targets involved in oxidative and inflammatory responses. While the in silico predictions are promising, further experimental validation is essential to confirm the binding affinities, target engagement, and pathway modulation in relevant biological systems.

3.8.3. Molecular Docking and Structure–Activity Relationship

To elucidate the molecular basis of the bioactivity of 5‐OHFA, molecular docking studies were performed on several key enzymes implicated in oxidative stress and inflammation. Comparative analyses with its parent compound, FA, enabled the identification of structural features responsible for enhanced binding and biological efficacy.

For NOX protein, 5‐OHFA demonstrated a stronger binding affinity (−6.1 kcal/mol) compared to FA (−5.9 kcal/mol) (Table 4). FA formed hydrogen bonds with SER326 and ILE160 and a π–σ interaction with LEU299. In contrast, 5‐OHFA established multiple additional interactions, including hydrogen bonds with THR9 and HIS79, a carbon–hydrogen bond with SER115, a π–sulfur interaction with MET33, and a π–anion interaction with GLU32, indicating greater binding stability (Figure 5).

FIGURE 5.

The 2D (a, b) and 3D (c, d) visualizations of intermolecular interactions established between NADPH oxidase protein (2CDU.pdb), toward FA (b, d) and 5‐OHFA (a, c), with binding energies of −5.9 and −6.1 kcal/mol, respectively.

Docking to XO revealed a notable difference: 5‐OHFA showed a binding energy of −7.0 versus −5.6 kcal/mol for FA. 5‐OHFA formed four hydrogen bonds (THR1083, ALA1079, and SER1080) and a C─H interaction with GLN1194. FA engaged fewer contacts, including two hydrogen bonds (VAL764 and LYS792) and a π–alkyl interaction (Figure 6), supporting the higher binding stability of 5‐OHFA.

FIGURE 6.

The 2D (a, b) and 3D (c, d) visualizations of intermolecular interactions established between xanthine oxidase protein (PDB: 1FIQ). Toward FA (b, d) and 5‐OHFA (a, c) with binding energies of −5.6 and −7 kcal/mol, respectively.

Both compounds bound effectively to COX‐2, with 5‐OHFA displaying binding energies ranging from −6.2 to −5.8 kcal/mol and FA between −7.0 and −5.6 kcal/mol. However, 5‐OHFA formed more diverse interactions, including four hydrogen bonds (GLY211, GLN360, ASN361 from two chains) and a π–π T‐shaped interaction, enhancing complex stability. FA exhibited fewer contacts, primarily hydrogen bonds with ASN361 and ARG362 (Figure 7).

FIGURE 7.

The 2D (a, b) and 3D (c, d) visualizations of intermolecular interactions established between cyclooxygenase‐2 (COX‐2) protein (PDB: 3LN1). Toward FA (b, d) and 5‐OHFA (a, c) with binding energies of −5.7 and −5.8 kcal/mol, respectively.

5‐OHFA formed three hydrogen bonds (TYR470 and GLN549) and π–alkyl interactions with VAL243 and ALA453. FA interacted with ARG370 and THR545 via hydrogen bonding, alongside a π–alkyl interaction. The enhanced number and diversity of interactions for 5‐OHFA suggest improved inhibitory potential against 5‐LOX (Figure 8).

FIGURE 8.

The 2D (a, b) and 3D (c, d) visualizations of intermolecular interactions established between 5‐LOX protein (PDB: 3O8Y). Toward FA (b, d) and 5‐OHFA (a, c), with binding energies of −6.2 and −6.1 kcal/mol, respectively.

5‐OHFA exhibited a binding energy of −5.0 kcal/mol, forming hydrogen bonds with SER191, ASN189, and MET190, and a π–σ interaction with ALA198. FA showed weaker binding (−4.8 kcal/mol), with fewer hydrogen bonds and a single π–alkyl interaction (Figure 9). These results suggest 5‐OHFA may more effectively modulate MPO activity, relevant in hemolysis and inflammation.

FIGURE 9.

The 2D (a, b) and 3D (c, d) visualizations of intermolecular interactions established between myeloperoxidase (MPO) protein (PDB: 1DNU). Toward FA (b, d) and 5‐OHFA (a, c) with binding energies of −4.8 and −5 kcal/mol, respectively.

Molecular docking was conducted to evaluate the binding interactions of the tested molecule, 5‐OHFA, and tamoxifen—a standard EGFR inhibitor—within the ATP‐binding site of the EGFR kinase domain (PDB ID: 1M17). This analysis aimed to elucidate the binding conformations and to provide molecular insights into the compounds' in vitro cytotoxic activity.

The binding affinities and detailed interaction profiles are summarized in Table 4. In silico docking results showed that 5‐OHFA had a binding energy of −5.9 kcal/mol, which is comparable to that of tamoxifen (−7.3 kcal/mol), consistent with previous findings by Triaa et al. [17]. Tamoxifen exhibited stable interactions through multiple hydrogen bonds—particularly with THR830—and π‐type interactions.

In the case of 5‐OHFA, binding within the EGFR active site was mediated by a rich and diverse interaction network, including four hydrogen bonds (with Lys721, Leu765, and two with Met769), two π–alkyl interactions (with Val702 and Ala719), a π‐donor hydrogen bond with Thr766, a carbon–hydrogen bond with Glu738, and several van der Waals contacts. Comparatively, FA exhibited a different binding pattern, involving three hydrogen bonds (ASN676, ALA678, and LEU736), π–alkyl interactions (ALA743 and LEU680), and a π–π stacking interaction with TYR740. Although both ligands displayed affinity for EGFR, 5‐OHFA established a more robust and diverse network of interactions, suggesting a superior binding potential over FA (Figure 10).

FIGURE 10.

The 2D (a, b) and 3D (c, d) visualizations of intermolecular interactions established between EGFR protein (PDB: 1M17). Toward FA (b, d) and 5‐OHFA (a, c) with binding energies of −5.8 and −5.9 kcal/mol, respectively.

Overall, the superior interaction profile and lower binding free energies of 5‐OHFA compared to FA across multiple enzymes are likely attributable to its additional hydroxyl group, which enhances hydrogen bonding and polar interactions. These molecular characteristics not only improve binding affinity but also contribute to the compound's dual antioxidant and anti‐inflammatory activity.

The docking results correlate well with in vitro findings, reinforcing a structure–activity relationship (SAR) wherein hydroxylation at the 5‐position plays a pivotal role in therapeutic potential. The ability of 5‐OHFA to engage robustly with catalytic residues of key targets supports its candidacy as a multifunctional agent in the development of novel anti‐inflammatory and antioxidant therapies.

4. Discussion

FA, well‐characterized hydroxycinnamic acid widely found in plant‐based foods, is renowned for its antioxidant, anti‐inflammatory, and anti‐cancer properties [34, 35]. Recent attention has turned to its hydroxylated derivative, 5‐OHFA, which exhibits enhanced bioactivity due to the addition of a hydroxyl group at the 5‐position of the aromatic ring [8]. This study combined in vitro biochemical assays and in silico analyses to comprehensively evaluate the pharmacological potential of 5‐OHFA, comparing its performance against FA across antioxidant, anti‐inflammatory, anti‐hemolytic, and anti‐proliferative activities.

The presence of an extra hydroxyl group at the 5‐position of the aromatic ring in 5‐OHFA significantly improves its radical‐scavenging capacity, as shown by DPPH, ABTS, and FRAP assays compared to FA. These results are consistent with previous reports indicating that hydroxylation increases electron‐donating ability [36], decreases bond dissociation enthalpy (BDE), and enhances resonance stabilization of phenoxyl radicals [37, 38]. Furthermore, the increased hydrophilicity of 5‐OHFA contributes to its solubility and accessibility to ROS in aqueous environments, further supporting its role as a potent antioxidant [8, 38].

Importantly, 5‐OHFA exhibited significant anti‐inflammatory activity, as demonstrated by its inhibitory effects on protein denaturation and hemolysis. In the BSA denaturation assay, 5‐OHFA exhibited a remarkably low IC₅₀ value of 15 µg/mL, indicating a potent capacity to preserve protein conformation under inflammatory conditions, in contrast to egg albumin (IC₅₀ = 125 µg/mL). This suggests a potential role in mitigating protein unfolding and aggregation processes involved in inflammatory pathogenesis. Since BSA denaturation under inflammatory conditions is closely associated with the exposure of hydrophobic residues and pro‐inflammatory signaling, the ability of 5‐OHFA to preserve protein structure suggests it could mitigate protein misfolding and aggregation—critical mechanisms in inflammation and oxidative stress‐induced damage. This protein‐stabilizing property underscores the compound's anti‐inflammatory potential through interaction with protein targets.

Consistently, 5‐OHFA exhibited strong anti‐hemolytic effects in human erythrocyte membrane stabilization assays, with an IC₅₀ of 29.53 ± 1.48 µM. These effects likely result from its interaction with membrane phospholipids and its ability to scavenge lipid peroxyl radicals, preserving erythrocyte integrity under oxidative stress. This aligns with known protective actions of polyphenols on cell membranes [23, 39]. Furthermore, molecular docking analysis supported the interaction of 5‐OHFA with MPO, an enzyme implicated in hemolysis. The compound formed hydrogen bonds and π–σ interactions with key residues (SER191, ASN189, and MET190), showing a favorable binding energy of −5.0 kcal/mol [10, 40].

The anti‐proliferative potential of 5‐OHFA was selectively evident in HCT116 colorectal carcinoma cells, with more modest effects observed on LS‐174T and MCF‐7 cell lines. This selective cytotoxicity may be attributed to differential cellular uptake or metabolism influenced by hydroxylation and hydrophilicity. Comparable behavior has been observed in structural analogs such as caffeic acid, known for its enhanced activity due to ortho‐dihydroxy substitution [41]. Notably, lipophilic derivatives of FA have demonstrated significantly enhanced cytotoxic activity against human breast cancer cells, emphasizing the critical role of hydrophobic modifications in modulating and potentially improving biological efficacy [42]. While the broad‐spectrum cytotoxicity of 5‐OHFA remains limited, its selectivity and low toxicity offer potential for targeted therapeutic applications, particularly in adjuvant chemotherapy. Structural optimization strategies (e.g., esterification or alkylation) may further potentiate its efficacy.

Molecular docking provided mechanistic insight into the superior performance of 5‐OHFA relative to FA. Docking simulations revealed consistently stronger binding affinities for 5‐OHFA across a panel of redox‐ and inflammation‐associated enzymes: NOX (−6.1 vs. −5.9 kcal/mol), XO (−7.0 vs. −6.8 kcal/mol), COX‐2 (−5.8 vs. −5.7 kcal/mol), 5‐LOX (−6.1 vs. −6.2 kcal/mol), MPO (−5.0 vs. −4.8 kcal/mol), and EGFR (−5.9 vs. −5.8 kcal/mol). These enhanced interactions were mediated by multiple types of bonds—hydrogen bonds, π–π stacking, van der Waals forces, and electrostatic contacts—with key active site residues such as THR9 and HIS79 (NOX), GLN360 (COX‐2), TYR470 (5‐LOX), and ALA198 (MPO) [43–45]. These molecular docking results corroborate the observed in vitro cytotoxic effects by highlighting the strong and specific interaction of 5‐OHFA with the ATP‐binding pocket of the EGFR kinase domain. The binding energy of 5‐OHFA (−5.9 kcal/mol), comparable to that of tamoxifen (−7.3 kcal/mol), along with its diverse interaction profile—including key hydrogen bonds with Met769 and Lys721 and hydrophobic contacts with residues such as Val702 and Ala719—suggests a stable and favorable binding mode. Compared to FA, 5‐OHFA established a more extensive and varied interaction network, which may account for its superior affinity and enhanced biological activity. These findings underscore the multi‐target potential of 5‐OHFA as an EGFR modulator and a promising therapeutic candidate.

The compound's structural features, notably the additional hydroxyl group, facilitate enhanced electron donation, resonance stabilization, and metal‐chelation—features that collectively improve its interaction with biological targets. SAR analysis further supports the notion that hydroxylation pattern significantly influences pharmacological efficacy [12, 46].

In silico ADMET predictions further corroborated the drug‐likeness of 5‐OHFA. Specifically, the calculated log P and TPSA values aligned well with the established physico‐chemical criteria for oral bioavailability, reinforcing its potential as a drug‐like compound [47, 48]. The molecule adheres to Lipinski's rule of five, exhibits high gastrointestinal absorption, favorable aqueous solubility, and limited BBB permeability—suggesting its potential for peripheral rather than CNS‐related applications [47, 48]. These pharmacokinetic attributes make 5‐OHFA suitable for both systemic and topical administration, especially where CNS exposure is undesirable.

Together, the in vitro and in silico findings provide robust evidence that 5‐OHFA is a multifunctional bioactive compound with potent antioxidant, anti‐inflammatory, anti‐hemolytic, membrane‐stabilizing, and selective anti‐proliferative properties. These biological activities are largely attributed to its structural features, notably the presence of an additional hydroxyl group at the 5‐position of the aromatic ring, which enhances its electronic reactivity and interaction with key protein targets. Furthermore, its favorable pharmacokinetic profile—including high gastrointestinal absorption, adequate solubility, and low toxicity—reinforces its potential for therapeutic development. Future research should prioritize in vivo validation, mechanistic elucidation, and structural optimization to fully realize its pharmacological utility.

5. Conclusion

The findings of this study highlight the strong antioxidant, anti‐inflammatory, and membrane‐stabilizing properties of 5‐OHFA, as demonstrated through a combination of in vitro biochemical assays and in silico modeling. 5‐OHFA exhibited multifaceted antioxidant activity—including radical scavenging, ferrous ion chelation, and reducing capacity—alongside significant anti‐inflammatory effects, as evidenced by its inhibition of protein denaturation and protection of erythrocyte membranes under oxidative stress. Molecular docking analyses further substantiated these results by revealing strong binding affinities for key oxidative and inflammatory enzymes such as NOX, XO, COX‐2, 5‐LOX, and MPO. In addition, in silico ADMET predictions confirmed favorable drug‐likeness, oral bioavailability, and a nontoxic profile, reinforcing its therapeutic potential. However, a key limitation of the current study is that comparative evaluations with FA were based on data extracted from the literature rather than direct experimental comparisons under identical conditions. Future studies will incorporate parallel testing with FA and other standard compounds to provide a more rigorous comparative framework.

To further validate the translational potential of 5‐OHFA, ongoing in vivo investigations are currently assessing its safety profile, pharmacokinetics, and organ‐specific efficacy, particularly in the brain, liver, and kidneys. These studies aim to define its therapeutic window and confirm its protective effects under physiological and pathophysiological conditions. Taken together, these findings support the continued development of 5‐OHFA as a promising multifunctional natural compound for pharmaceutical or nutraceutical applications targeting oxidative stress and inflammation‐related disorders, including neurodegenerative diseases, metabolic syndromes, and toxin‐induced organ damage.

Ethics Statement

All procedures involving the collection and use of animal blood were conducted in accordance with institutional and international ethical guidelines. The study received formal approval from the Animal Ethics Committee of the National School of Veterinary Medicine, Sidi Thabet, Tunisia (approval number: CEEA‐ENMV 70/23, dated March 26, 2024).

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Sakouhi S., Ben Younes S., Arrari F., Nahdi A., Bouajila J., and Mhamdi A., “Integrated In Vitro and In Silico Characterization of 5‐Hydroxyferulic Acid: Antioxidant, Anti‐Inflammatory, Anti‐Hemolytic, and Cytotoxic Potential.” Chemistry & Biodiversity 22, no. 12 (2025): e01431. 10.1002/cbdv.202501431

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.